Use of sarmentine and its analogs for controlling plant pests

ABSTRACT

Methods and compositions for controlling plant pests, particularly weeds and/or plant phytopathogens using sarmentine and/or analogs thereof are disclosed.

FIELD OF THE INVENTION

This invention relates to compositions and methods for controlling plantpests, particularly weeds and/or plant phytopathogens such as plantpathogenic bacteria, viruses, fungi, nematodes, and insects usingsarmentine or analogs thereof as an active ingredient.

BACKGROUND OF THE INVENTION

Utilization of synthetic herbicides not only prevents economic loss infood production, but also improves quality of crop products (Gianessi,L. P.; Reigner, N. P. The value of herbicides in U.S. crop production,Weed Technol. 2007, 21, 559-566). However, the use of syntheticherbicides may cause side-effects on environment and human health(Solomon, G. M.; and Schettler, T. Environment and health: 6. Endocrinedisruption and potential human health implications, CMAJ, 2000, 163,1471-1476; Stillerman, K. P.; Mattison, D. R.; Giudice, L. C.; Woodruff,T. J. Environmental exposures and adverse pregnancy outcomes: A reviewof the science. Reproductive Sci. 2008, 15, 631-650) except that it alsoleads to an increasing resistance among many weed species (Whaley, C.M.; Wilson, H. P.; Westwood, J. H. A new mutation in plant ALS confersresistance to five classes of ALS-inhibiting herbicides, Weed Sci. 2007,55, 83-90). Therefore, it is very necessary to develop alternative meansfor weed management that are eco-friendly, cost-effective andbio-efficacious (Duke, S. O.; Dayan, F. E.; Rimando, A. M.; Schrader, K.K.; Aliotta, G.; Oliva, A.; Romagni, J. G. Chemicals from nature forweed management. Weed Sci. 2002, 50, 138-151).

The use of natural phytochemicals is one of these alternative means(Batish, D. R.; Setia, N.; Singh, H. P.; Kohli, R. K. Phytotoxicity oflemon-scented eucalypt oil and its potential use as a bioherbicide, CropProtection, 2004, 23, 1209-1214). Due to allelopathic properties, manyof these chemicals are either released in air or soil to killneighboring weeds or to inhibit their germination and/or growth. Thesephytotoxic chemicals include phenolic compounds (e.g., catechin, ellagicacid, sorgoleone, juglone, ceratiolin, usnic acid), terpenoids (e.g.,1,8-cineole, geranial, neral, cinmethylin, solstitiolide), quassinoids(e.g., ailanthone, chaparrine, ailanthinol B), benzoxazinoids (e.g.,hydroxamic acids), glucoinolates (e.g., glucohirsutin, hirsutin,arabin), and some amino acids such as meta-tyrosine (Macias, F. A.;Molinillo, J. M. G.; Varela, R. M.; Galindo, J. C. J. Allelopathy—anatural alternative for weed control. Pest Manag Sci., 2007, 63,327-348; Bertin, C.; Weston, L. A.; Huang, T.; Jander, G.; Owens, T.;Meinwald, J.; Schroeder, F. C. Grass roots chemistry: meta-Tyrosine, anherbicidal nonprotein amino acid. PNAS, 2007, 104, 16964-16969).Commercially available herbicides include products based on clove oil,lemongrass oil and d-limonene.

The Genus Piper

The genus Piper in the Piperaceae family contains approximately 2000species found primarily in tropical regions. Plants of this genus arenormally slender aromatic climbers with perennial woody roots. Thefruits commonly known as “pippali” in India and “Bi Bo” in China areused as a spice and also as a preservative in pickles. They are alsoused as cattle feed.

In traditional medicinal practice, P. longum fruits have been advocatedto be beneficial in treatment of diseases such as gonorrhea, menstrualpain, tuberculosis, sleeping problems, respiratory tract infections,chronic gut-related pain, and arthritic conditions (Krishnamurthi, A.1969. The Wealth of India Raw Materials, vol. 8. CSIR, New Delhi, India,p. 96; Ghoshal, S.; Prasad, B. N. K.; Lakshmi, V. Antiamoebic activityof Piper longum fruits against Entamoeba histolytica in vitro and invivo, J. Ethanopharmacol. 1996, 50, 167-170; Choi, E. M.; Hwang, J. K.Investigations of anti-inflammatory and antinociceptive activities ofPiper cubeba, Physalis angulata and Rosa hybrid, J. Ethanopharmacol.2003, 89, 171-175, Mata, R.; Morales, I.; Perez, O.; Rivero-Cruz, I.;Acevedo, L.; Enriquez-Mendoza, I.; Bye, R.; Franzblau, S.; Timmermann,B. Antimycobacterial compounds from Piper sanctum, J. Nat. Prod. 2004,67, 1961-1968). Other reported beneficial effects of P. longum includeanalgesic and diuretic effects, relaxation of muscle tension, andalleviation of anxiety (Vedhanayaki, G.; Shastri, G. V.; Kuruvilla, A.Analgesic activity of Piper longum Linn. Root, Ind. J. Exp. Biol. 2003,41, 649-651; Das, Biswanath, D.; Kashinatham, A.; Srinivas, K. V. N. S.Alkamides and other constituents of Piper longum, Planta Med, 1996, 62,582). In addition, pipernonaline from P. longum has been found topossess mosquito larvicidal activity (Yang, Y. C.; Lee, S. G.; Lee, H.K.; Kim, M. K.; Lee, S. H.; Lee, H. S. A piperidine amide extracted fromPiper longum L. fruit shows activity against Aedes aegypti MosquitoLarvae, J. Agric. Food Chem., 2002, 50, 3765-3767).

An alkaloid has been isolated from Piper nigrum (pepper) which is analkenylene piperidine amide containing a C18 alkenylene with two or moredouble bonds. This compound has been found to inhibit mycotoxinbiosynthesis. (U.S. Pat. No. 6,825,216).

Sarmentine

N-(2E,4E-Decadienoyl)pyrrolidine (also called sarmentine) was originallyseparated from the fruit of Piper sarmentosum in 1987 [Likhitwitayawuid,K., Ruangrungsi, N, Lange, G. and Decicco, C., Structural Elucidationand Synthesis of New Components isolated from Piper Samentosum,Tetrahedron 1987 (43) 3689-3694] and also from Piper nigrum in 1988[Kiuchi, F., Nakamura, N., Tsuda, Y., Kondo, K. and Yoshimura, H.Studies on Crude Drugs Effective on Visceral Larva Migrans. IV.Isolation and Identification of Larvicidal Principles in Pepper Chemicaland Pharmaceutical Bulletin 1988 (36):2452], and first synthesized in1995 [Bernabeu, M., Chinchilla, R. and Najera, C.,(2E,4E)-5-Tosyl-2,4-pentadienamides: New Dienic Sulfones for theStereoselective Synthesis of (2E,4E)-Dienamides, Tetrahedron Letter,1995 (36) 3901-3904]. Sarmentine has been found to be in vivo skinantioxidant protecting photoaged skin [Cornacchione, S.; Sadick, N. S.;Neveu, M.; Talbourdet, S.; Lazou, K.; Viron, C.; Renimel, I.; de Quéral,D.; Kurfurst, R.; Schnebert, S.; Heusèle, C.; André, P.; Perrier E. Invivo skin antioxidant effect of a new combination based on a specificVitis vinifera shoots extract and a biotechnological extract. J. Drugsin Dermatol. 2007, 6S, 8-13], display antiplatelet aggregation activity[Li, C. Y.; Tsai, W.; Damu, A. G.; Lee, E. J.; Wu, T. S.; Dung. N. X.;Thang, T. D.; Thanh, L. Isolation and identification of antiplateletaggregatory principles from the leaves of Piper lolot, J. Agric. FoodChem. 2007, 55, 9436-9442], antiplasmodial and antimycobacterialactivities [Tuntiwachwuttikul, P.; Phansa, P.; Pootaeng-on, Y.; Taylor,W. C. Chemical constituents of the roots of Piper Sarmentosum, Chem.Pharm. Bull. 2006, 54, 149-151] and antituberculosis activity[Rukachaisirikul, T.; Siriwattanakit, P.; Sukcharoenphol, K.; Wongvein,C.; Ruttanaweang, P.; Wongwattanavuch, P.; Suksamrarn, A. Chemicalconstituents and bioactivity of Piper sarmentosum, J. Ethnopharmacol.,2004, 93, 173-176]. Sarmentine is used as a solubilizer of hydrophobiccompounds in cosmetics and pharmaceuticals (Stephen, T.; Andrew, H.Compositions comprising macromolecular assembles of lipid surfactant,PCT Publication No. WO/2008/065451).

BRIEF SUMMARY OF THE INVENTION

The invention is directed to compositions comprising sarmentine and/orits analogs for use against plant pests, particularly plantphytopathogens such as plant pathogenic bacteria, fungi, insects,nematodes and/or as a pre- and post-emergence herbicide against weeds,as well as the use of sarmentine and/or its analogs in formulating suchpesticidal (phytopathogenic or herbicidal composition). In a particularembodiment, the sarmentine analog(s) which may be used in compositionsand methods of the present invention has substantially the same activityas sarmentine. As defined herein “substantially the same activity assarmentine” means that it has at least about 80% of the phytopathogenicand/or herbicidal activity of sarmentine and preferably at least about90% of the phytopathogenic and/or herbicidal activity of sarmentine andeven more preferably at least about 95% of the phytopathogenic and/orherbicidal activity of sarmentine.

The sarmentine analogs includes but is not limited to:N-(Decanoyl)pyrrolidine, N-(Decenoyl)pyrrolidine,N-(Decanoyl)piperidine, N-(trans-Cinnamoyl)pyrrolidine,(2E,4Z-Decadienoyl)pyrrolidine, N-(Decenoyl)piperidine,(2E,4Z-Decadienoyl)piperidine, (2E,4Z-Decadienoyl)hexamethyleneimine,N-(Decenoyl)hexamethyleneimine, N-(Decanoyl)hexamethyleneimine, decanoicacid, 2E-Decenoic acid.

In a particular embodiment, the invention is directed to an isolatedsarmentine analog having the structure:

Where R1 is an alkyl, alkenyl, alkynyl, heterocyclic, aromatic, arylgroup, NH-substituted, or N,N-substituted group, the length of R1 chaincan be from 4 to 20 atoms, the preferred length will be from 6 to 12atoms;Wherein R2 and R3 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,aromatic, arylalkyl, heterocyclyl or heteroaryl or R2+R3+N can be anN-containing heterocyclic or herteroaryl ring consisting of between 3-18atoms and preferably between 5 to 8 atoms.

In a more particular embodiment, the isolated samentine analog includesbut is not limited to: (2E,4Z-Decadienoyl)pyrrolidine;(2E,4Z-Decadienoyl)hexamethyleneimine andN-(Decenoyl)hexamethyleneimine.

The compositions of the present invention may further comprise one ormore other herbicides or phytopathogenic modulating agents. Theinvention is also directed to uses of one or more sarmentine and/or itsanalogs for preparation of a composition for use as a pre or postemergent herbicide and/or as an anti-phytopathogenic agent.

In a particular embodiment, the invention is directed to a method formodulating phytopathogenic infection in a plant comprising applying tothe plant and/or seeds thereof and/or substrate used for growing saidplant an amount of a sarmentine and/or its analogs effective to modulatesaid phytopathogenic infection. The substrate for growing said plant ina particular embodiment may include but is not limited to soil, anartificial growth substrate, water or sediment.

In another particular embodiment, the invention is directed to a methodfor modulating emergence of monocotyledonous, or dicotyledonous weeds ina substrate comprising applying to the weeds and/or substrate an amountof a sarmentine and/or its analogs effective to modulate emergence ofmonocotyledonous or dicotyledonous weeds in the substrate. The substratemay include but is not limited to soil, an artificial growth substrate(e.g., rice growing system), water or sediment. The sarmentine and/orits analogs is applied to the substrate prior to emergence of said weed.Alternatively, the sarmentine and/or its analogs may be applied to thesubstrate and/or weed after emergence of said weed(s).

The weeds may be broadleaved and/or grass weeds. In a particularembodiment, the weeds are in a rice growing system and the weed is arice weed(s).

In a particular embodiment, the sarmentine and/or its analogs is appliedin an amount of about 0.005 mg/ml to about 20 mg/ml. In a moreparticular embodiment, the sarmentine and/or its analogs is applied inan amount of about 0.01 to about 15 mg/ml. In yet a more particularembodiment, the sarmentine and/or its analogs is applied in an amount ofabout 0.1 to about 10 mg/ml.

DETAILED DESCRIPTION OF THE INVENTION

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

As defined herein, the term “modulate” is used to mean to alter theamount of phytopathogenic infection or rate of spread of phytopathogenicinfection.

Sarmentine and its Analogs

The sarmentine and/or its analogs used in the method of the presentinvention may have the following structure:

Where X includes but is not limited to sulfur, phosphorus, boron orcarbon; Y includes but is not limited to carbon, oxygen, nitrogen,sulfur, boron or phosphorous; R₁ includes but is not limited tohydrogen, hydroxyl, halogen, alkyl, alkoxy, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aromatic, arylalkyl, heterocyclic andheteroaryl; R₂ includes but is not limited to hydrogen, hydroxyl,halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aromatic,arylalkyl, heterocyclyl and heteroaryl; R₃ includes but is not limitedto hydrogen, hydroxyl, halogen, alkyl, alkoxy, alkenyl, alkynyl,cycloalkyl, aromatic, arylalkyl, heterocyclic and heteroaryl; whereinR₂+R₃+Y can be a cyclic or heterocyclic ring containing 4-50 atoms. Eachof these is optionally substituted.

As used herein, the term “alkyl” refers to a saturated hydrocarbonradical which may be straight-chain or branched-chain (e.g., ethyl,isopropyl, t-amyl, or 2,5-dimethylhexyl, etc.). This definition appliesboth when the term is used alone and when it is used as part of acompound term.

The terms “cycloalkyl” and “cycloalkenyl” refer to a saturatedhydrocarbon ring and includes bicyclic and polycyclic rings. Similarly,cycloalkyl and cycloalkenyl groups having a heteroatom (e.g., N, O, orS) in place of a carbon ring atom may be referred to as“heterocycloalkyl”, “heterocyclyl,” and “heterocycloalkylene,”respectively.

The term “alkenyl” as used herein refers to an alkyl group as describedabove which contains one or more sites of unsaturation that is a doublebond. Similarly, the term “alkynyl” as used herein refers to an alkylgroup as described above which contains one or more sites ofunsaturation that is a triple bond.

The term “alkoxy” refers to an alkyl radical as described above whichalso bears an oxygen substituent which is capable of covalent attachmentto another hydrocarbon radical (such as, for example, methoxy, ethoxy,aryloxy, and t-butoxy).

The term “aryl” refers to an aromatic carbocyclic substituent which maybe a single ring or multiple rings which are fused together, linkedcovalently or linked to a common group such as an ethylene or methylenemoiety. Similarly, aryl groups having a heteroatom (e.g., N, O, or S) inplace of a carbon ring atom are referred to as “heteroaryl.”

The terms “arylalkyl,” “arylalkenyl,” and “aryloxyalkyl” refer to anaryl radical attached directly to an alkyl group, an alkenyl group, oran oxygen atom which is attached to an alkyl group, respectively. Forbrevity, aryl as part of a combined term as above is meant to includeheteroaryl as well.

The term “halo” or “halogen,” by itself or as part of anothersubstituent, means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl.

The term “hetero” as used in a “heteroatom-containing alkyl group”(i.e., a “heteroalkyl” group) or a “heteroatom-containing aryl group”(i.e., a “heteroaryl” group) refers to a molecule, linkage, orsubstituent in which one or more carbon atoms are replaced with an atomother than carbon, e.g., nitrogen, oxygen, sulfur, phosphorus, orsilicon.

In one particular embodiment, the sarmentine analog has the followingstructure:

Where R₁ is an alkyl, alkenyl, alkynyl, herterocyclyl, aromatic, arylgroup, NH-substituted, or N,N-substituted group. In a specificembodiment, R₁ is an alkyl or alkenyl moiety containing from 4 to 20atoms and preferably from 6 to 12 atoms. In a more specific embodiment,R₁ is a C₅₋₁₅ alkyl or C₅₋₁₅ alkenyl group. In yet a very specificembodiment, R₁ is a C₆₋₁₂ alkyl or C₆₋₁₂ alkenyl group. Possible alkenylinclude but are not limited to linear alkenyl fatty acids, branchedalkenyl fatty acids, cycloalkenyl substituted fatty acids (e.g.,cyclohexenylpropanoic acid, cyclohexenylbutanoic acid,cyclohexenylpentanoic acid and so on), heterocycloalkenyl (e.g.,4-[1,2,3,4-tetrahydropyridinyl] butanoic acid).

In another particular embodiment, the sarmentine analog has thefollowing structure:

Where R1 is an alkyl, alkenyl, alkynyl, heterocyclic, aromatic, arylgroup, NH-substituted, or N,N-substituted group, the length of R₁ chaincan be from 4 to 20 atoms, the preferred length will be from 6 to 12atoms.Wherein R₂ and R₃ are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,aromatic, arylalkyl, heterocyclic or heteroaryl; or alternativelyR₂+R₃+N can be an N-containing heterocyclic moiety.

One of skill in the art will appreciate that any sarmentinederivatives-containing materials for weed or phytopathogen management isincluded. Sarmentine derivatives in these materials can be naturaland/or synthesized.

In certain embodiments, natural sarmentine derivatives may be obtainedfrom plants, fungi, bacteria and soils. In a particular embodiment,sarmentine and its analogs used in the method of the present inventionmay be obtained from the fruits, leaves, stems and roots of any Piperspecies. In a more particular embodiment, non-limiting examples of Piperspecies that may contain sarmentine derivatives include but are notlimited to the following species such as Piper aborescens, P.acutisleginum, P. aduncum, P. amalago, P. argyrophylum, P. attenuatum,P. auranticaum, P. austrosinense T., P. arboricola C. DC., P. banksii,P. bartlingianum, P. betle L., P. boehmeriifolium var. tonkinense C.DC., P. brachystachyum, P. callosum, P. chaba, P. chiadoense, P. cubebaL., P. damiaoshaneense, P. demeraranum, P. falconeri, P. futokadsura, P.guayranum, P. guineense, P. hainanense Hemsl. in F. B. Forbes andHemsl., P. hamiltonii, P. hancei Maxim., P. khasiana, P. kadsura(Choisy) Ohwi, P. laetispicum C. DC., P. longum L., P. longum var.(“round peepal”), P. macropodum, P. manii, P. martinii C. DC., P.methysticum, P. nepalense, P. novae hollandiae, P. nigrum L., P.nudibaccatum Y. C. Tseng, P. officinarum, P. peepuloides, P.pedicellosum, P. ponesheense C. DC., P. puberulilimbum C. DC., P.puberulum (Benth.) Maxim., P. pubicatulum C. DC., P. ridleyi, P.rugosum, P. retrofractum Vahl, P. ribesioides, P. sanctum, P.sarmentosum R., P. schmidtii, P. semiimmersum C. DC., P. sintenense, P.spirei C. DC., P. syvaticum, P. thomsoni, P. verruscosum, P.trichostachyon, P. wallichii (Miq.), P. wightii [Parma, V., Jain, S.,Bisht, K., Jain, R., Poonam, T., Jha, A., Tyagi, O., Prasad, A., Wengel,J., Olsen, C. and Boll., P., Phytochemistry of the Genus Piper,Phytochem. 1997 (46) 597-673; Parma, V., Jain, S., Gupta, S., Talwar,S., Rajwanshi, V., Kumar, R., Azim, A., Malhotra, S., Kumar, N., Jain,R., Sharma, N., Tyagi, O., Lawrie, S., Errington, W., Howarth, O.,Olsen, C., Singh, S, and Wengel, J. Polyphenols and Alkaloids from PiperSpecies Phytochem. 1998 (49) 1069-10781. Sarmentine derivatives may alsobe obtained from microorganisms such as Actinomycetes [Cho, J.,Williams, P., Kwon, H., Jensen, P., and Fenical, W., Lucentamycins A-D,Cytotoxic Peptides form the Marine-Derived Actinomycete Nocardiopsislucentensis, J. Nat. Prod., 2007 (70) 1321-1328; Askolar, R., Jensen,P., Kauffman, C., and Fenical, W., Daryamides A-C, Weakly CytotoxicPolyketides form a Marine-Derived Actinomycete of the Genus StreptomycesStrain CNQ-085, J. Nat. Prod., 2006 (69), 1756-1759].

Sarmentine derivatives can be extracted and purified in any physical andchemical means from Piper longum using procedures set forth in theExample, infra, or using procedures known in the art (see, for example,Likhitwitayawuid, K., Ruangrungsi, N, Lange, G. and Decicco, C.,Structural Elucidation and Synthesis of New Components isolated fromPiper Samentosum Tetrahedron 1987 (43) 3689-3694 and Kiuchi, F.,Nakamura, N., Tsuda, Y., Kondo, K. and Yoshimura, H. Studies on CrudeDrugs Effective on Visceral Larva Migrans. IV. Isolation andIdentification of Larvicidal Principles in Pepper Chemical andPharmaceutical Bulletin 1988 (36):24521. They can also be chemicallysynthesized using for example, the method set forth in [Bernabeu, M.,Chinchilla, R. and Najera, C., (2E,4E)-5-Tosyl-2,4-pentadienamides: NewDienic Sulfones for the Stereoselective Synthesis of (2E,4E)-Dienamides,Tetrahedron Letter, 1995 (36) 3901-]]. In a particular embodiment, aPiper longum sample is subject to extraction with an alkyl alcohol,preferably methanol. Sarmentine is subsequently isolated from theextract by for example, column chromatography, more particularly by HPLCand fractions containing the sarmentine are identified by, for examplebioassay.

In a particular embodiment, the compound used may be sarmentine, alsoknown as N-(2E,4E-decadienoyl)pyrrolidine. Natural sarmentine can existin either plant extracts or a purified form. The sarmentine analog mayalso be N-(Decanoyl)pyrrolidine, N-(Decenoyl)pyrrolidineN-(Decanoyl)piperidine, N-(trans-Cinnamoyl)pyrrolidine,(2E,4Z-Decadienoyl)pyrrolidine N-(Decenoyl)piperidine,(2E,4Z-Decadienoyl)piperidine, (2E,4Z-Decadienoyl)hexamethyleneimine,N-(Decenoyl)hexamethyleneimine, N-(Decanoyl)hexamethyleneimine, Decanoicacid or 2E-Decenoic acid.

Formulations

Sarmentine and/or sarmentine analog-containing herbicidal compositions(also alternatively referred to as “formulations”) can be formulated inany form. Non-limiting formulation examples include emulsifiableconcentrates (EC), wettable powders (WP), soluble liquids (SL),aerosols, ultra-low volume concentrate solutions (ULV), soluble powders(SP), microencapsulation, water dispersed granules, flowables (FL),microemulsions (ME), nano-emulsions (NE), etc. In any formulationdescribed herein, percent of sarmentine and/or its analogs is within arange of 0.01% to 99.99%. In a particular embodiment, the formulationsmay be free of surfactants.

The compositions of the invention may further comprise a carrier and/ordiluent. The term, ‘carrier’ as used herein means an inert, organic orinorganic material, with which the active ingredient is mixed orformulated to facilitate its application to the soil, seed, plant orother object to be treated, or its storage, transport and/or handling.Examples of carrier vehicles to be used when applying to growthsubstrates include, but are not limited to, active charcoal, corn glutenmeal, soybean meal, vermiculite, bentonite, kaolinite, wheat germ,almond hulls, cottonseed meal, Fuller's earth, orange pulp, rice hulls,sawdust, Gum arabic, etc. If desired, plant essential oils such ascinnamon, clove, thyme (eugenol as active ingredient), wintergreen, soymethyl ester, citronella and pine oil, citrus oil (1-limonene as activeingredient) and the like, can be included in the granules. As notedabove, the active ingredient alone or in the presence of the carriervehicles, may be dissolved in for example, water, or organic solventsuch as ethanol, formic acid or ethanol.

In further embodiments, sarmentine and its analogs themselves are easilyoxidized because of two conjugated double bonds. This is proven by thefact that sarmentine can be an in vivo antioxidant for photoaged skin 11[Cornacchione, S.; Sadick, N. S.; Neveu, M.; Talbourdet, S.; Lazou, K.;Viron, C.; Renimel, I.; de Queral, D.; Kurfurst, R.; Schnebert, S.;Heusele, C.; Andre, P.; Perrier, E., In vivo skin antioxidant effect ofa new combination based on a specific Vitis vinifera shoot extract and abiotechnological extract, J. drugs in Dermatol. 2007 (6 suppl) S8-13].Therefore, any antioxidant can be added into the sarmentine and/or itsanalogs-containing formulation to boost and/or elongate the phytotoxicactivity. The non-limiting examples of antioxidants include alphatocopherol, beta carotene, ascorbic acid, zinc oxide, titanium oxide,Gynostemma pentaphyllum extract, Vaccinium angustifolium (Blueberry)fruit extract, Pinus strobus bark extract, rhaponticin, planktonextract, Monostroma sp. extract, algae extract, venuceane, rosmarinicacid, and any other plant extracts or antioxidants.

Examples of phytopathogens controlled by sarmentine and/or its analogsin the method of the present invention include but are not limited toplant viruses, phytopathogenic fungi or bacteria, insects or nematodes.In a specific embodiment, the viruses include but are not limited toTMV, tobacco or cucumber mosaic virus, ringspot virus, necrosis virus,maize dwarf mosaic virus. Phytopathogenic fungi include but are notlimited to Fusarium sp., Botrytis sp., Monilinia sp., Colletotrichum sp,Verticillium sp.; Microphomina sp., and Phytophtora sp., Mucor sp.,Rhizoctonia sp., Geotrichum sp., Phoma sp., and Penicillium sp.Phytopathognic bacteria include but is not limited to Bacillus sp. orXanthomonas sp.

Nematodes that may be controlled using the method of the presentinvention include but are not limited to parasitic nematodes such asroot-knot, cyst, and lesion nematodes, including Heterodera andGlobodera sp.; particularly Globodera rostochiensis and G. pailida(potato cyst nematodes); Heterodera glycines (soybean cyst nematode); H.schachtii (beet cyst nematode); and H. avenae (cereal cyst nematode).

Phytopathogenic insects controlled by the method of the presentinvention include but are not limited to insects from the order (a)Lepidoptera, for example, Acleris sp., Adoxophyes sp., Aegeria sp.,Agrotis sp., Alabama argillaceae, Amylois sp., Anticarsia gemmatalis,Archips sp., Argyrotaenia sp., Autographa sp., Busseola fusca, Cadracautella, Carposina nipponensis, Chilo sp., Choristoneura sp., Clysiaambiguella, Cnaphalocrocis sp., Cnephasia sp., Cochylis sp., Coleophorasp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydia sp.,Diatraea sp., Diparopsis castanea, Earias sp., Ephestia sp., Eucosmasp., Eupoecilia ambiguella, Euproctis sp., Euxoa sp., Grapholita sp.,Hedya nubiferana, Heliothis sp., Hellula undalis, Hyphantria cunea,Keiferia lycopersicella, Leucoptera scitella, Lithocollethis sp.,Lobesia botrana, Lymantria sp., Lyonetia sp., Malacosoma sp., Mamestrabrassicae, Manduca sexta, Operophtera sp., Ostrinia nubilalis, Pammenesp., Pandemis sp., Panolis flammea, Pectinophora gossypiella,Phthorimaea operculella, Pieris rapae, Pieris sp., Plutella xylostella,Prays sp., Scirpophaga sp., Sesamia sp., Sparganothis sp, Spodoptera sp,Synanthedon sp., Thaumetopoea sp., Tortrix sp., Trichoplusia ni andYponomeuta sp.; (b) Coleoptera, for example, Agriotes sp., Anthonomussp., Atomaria linearis, Chaetocnema tibialis, Cosmopolites sp., Curculiosp., Dermestes sp., Diabrotica sp., Epilachna sp., Eremnus sp.,Leptinotarsa decemlineata, Lissorhoptrus sp., Melolontha sp.,Orycaephilus sp., Otiorhynchus sp., Phlyctinus sp., Popillia sp.,Psylliodes sp., Rhizopertha sp-, Scarabeidae, Sitophilus sp., Sitotrogasp., Tenebrio sp., Tribolium sp. and Trogoderma sp.; (c) Orthoptera, forexample, Blatta sp., Blattella sp., Gryllotalpa sp., Leucophaea maderae,Locusta sp., Periplaneta sp. and Schistocerca sp.; (d) Isoptera, forexample, Reticulitermes sp.; (e) Psocoptera, for example, Liposcelissp.; (f) Anoplura, for example, Haematopinus sp., Linognathus sp.,Pediculus sp., Pemphigus sp. and Phylloxera sp.; (g) Mallophaga, forexample, Damalinea sp. and Trichodectes sp.; (h) Thysanoptera, forexample, Frankliniella sp., Hercinotnrips sp., Taeniothrips sp., Thripspalmi, Thrips tabaci and Scirtothrips aurantii; (i) Heteroptera, forexample, Cimex sp., Distantiella theobroma, Dysdercus sp., Euchistussp., Eurygaster sp., Leptocorisa sp., Nezara sp., Piesma sp., Rhodniussp., Sahlbergella singularis, Scotinophara sp. and Tniatoma sp.; (j)Homoptera, for example, Aleurothrixus floccosus, Aleyrodes brassicae,Aonidiella sp., Aphididae, Aphis sp., Aspidiotus sp., Bemisia tabaci,Ceroplaster sp., Chrysomphalus aonidium, Chrysomphalus dictyospermi,Coccus hesperidum, Empoasca sp., Eriosoma larigerum, Erythroneura sp.,Gascardia sp., Laodelphax sp., Lecanium corni, Lepidosaphes sp.,Macrosiphus sp., Myzus sp., Nephotettix sp., Nilaparvata sp., Paratoriasp., Pemphigus sp., Planococcus sp., Pseudaulacaspis sp., Pseudococcussp., Psylla sp., Pulvinaria aethiopica, Quadraspidiotus sp.,Rhopalosiphum sp., Saissetia sp., Scaphoideus sp., Schizaphis sp.,Sitobion sp., Trialeurodes vaporariorum, Trioza erytreae and Unaspiscitri; (k) Hymenoptera, for example, Acromyrmex, Atta sp., Cephus sp.,Diprion sp., Diprionidae, Gilpinia polytoma, Hoplocampa sp., Lasius sp.,Monomorium pharaonis, Neodiprion sp., Solenopsis sp. and Vespa sp.; (1)Diptera, for example, Aedes sp., Antherigona soccata, Bibio hortulanus,Calliphora erythrocephala, Ceratitis sp., Chrysomyia sp., Culex sp.,Cuterebra sp., Dacus sp., Drosophila melanogaster, Fannia sp.,Gastrophilus sp., Glossina sp., Hypoderma sp., Hyppobosca sp., Liriomyzasp., Lucilia sp., Melanagromyza sp., Musca sp., Oestrus sp., Orseoliasp., Oscinella frit, Pegomyia hyoscyami, Phorbia sp., Rhagoletispomonella, Sciara sp., Stomoxys sp., Tabanus sp., Tannia sp. and Tipulasp.; (m) Siphonaptera, for example, Ceratophyllus sp. and Xenopsyllacheopis and (n) from the order Thysanura, for example, Lepismasaccharina. The active ingredients according to the invention mayfurther be used for controlling crucifer flea beetles (Phyllotreta sp.),root maggots (Delia sp.), cabbage seedpod weevil (Ceutorhynchus sp.) andaphids in oil seed crops such as canola (rape), mustard seed, andhybrids thereof, and also rice and maize. Sarmentine and/or its analogsin these materials can be the only active ingredient(s) or a mixturewith any other phytopathogenic and/or herbicidal compounds. In someembodiments, non-limiting examples of natural herbicides that can usedwith sarmentine and its analogs include but are not limited to catechin,ellagic acid, sorgoleone, juglone, ceratiolin, usnic acid, 1,8-cineole,geranial, neral, cinmethylin, solstitiolide, ailanthone, chaparrine,ailanthinol B, hydroxamic acids, glucohirsutin, hirsutin, arabin,meta-tyrosine. Percent of sarmentine and/or its analogs in thesecompositions can be within a range of 0.01% to 99.99%.

In other embodiments, non-limiting examples of synthetic herbicides thatcan used with sarmentine and/or its analog include but are not limitedto aryloxyphenoxypropionic herbicides (e.g., chlorazifop, clodinafop,clofop, cyhalofop, diclofop, fenoxaprop, fenoxaprop-P, fenthiaprop,fluazifop, fluazifop-P, haloxyfop, haloxyfop-P, isoxapyrifop, metamifop,propaquizafop, quizalofop, quizalofop-P and trifop); benzoic acidherbicides (e.g., chloramben, dicamba, 2,3,6-TBA and tricamba);benzofuranyl alkylsulfonate herbicides (e.g., benfuresate andethofumesate); benzoylcyclohexanedione herbicides (e.g., mesotrione,sulcotrione, tefuryltrione and tembotrione); carbamate herbicides (e.g.,asulam, carboxazole chlorprocarb, dichlormate, fenasulam, karbutilateand terbucarb); carbanilate herbicides (e.g., barban, BCPC, carbasulam,carbetamide, CEPC, chlorbufam, chlorpropham, CPPC, desmedipham,phenisopham, phenmedipham, phenmedipham-ethyl, propham); cyclohexeneoxime herbicides (e.g., alloxydim, butroxydim, clethodim, cloproxydim,cycloxydim, profoxydim, sethoxydim, tepraloxydim and tralkoxydim);cyclopropylisoxazole herbicides (e.g., isoxachlortole and isoxaflutole);dicarboximide herbicides (e.g., benzfendizone, cinidon-ethyl, flumezin,flumiclorac, flumioxazin and flumipropyn); dinitroaniline herbicides(e.g., benfluralin, butralin, dinitramine, ethalfluralin, fluchloralin,isopropalin, methalpropalin, nitralin, oryzalin, pendimethalin,prodiamine, profluralin and trifluralin); dinitrophenol herbicides(e.g., dinofenate, dinoprop, dinosam, dinoseb, dinoterb, DNOC, etinofenand medinoterb); dithiocarbamate herbicides (e.g., dazomet and metam;halogenated aliphatic herbicides such as alorac, chloropon, dalapon,flupropanate, hexachloroacetone, iodomethane, methyl bromide,monochloroacetic acid, SMA and TCA); imidazolinone herbicides (e.g.,imazamethabenz, imazamox, imazapic, imazapyr, imazaquin andimazethapyr); inorganic herbicides (e.g., ammonium sulfamate, borax,calcium chlorate, copper sulfate, ferrous sulfate, potassium azide,potassium cyanate, sodium azide, sodium chlorate and sulfuric acid);nitrophenyl ether herbicides (e.g., acifluorfen, aclonifen, bifenox,chlomethoxyfen, chlornitrofen, etnipromid, fluorodifen, fluoroglycofen,fluoronitrofen, fomesafen, furyloxyfen, halosafen, lactofen, nitrofen,nitrofluorfen and oxyfluorfen); nitrile herbicides (e.g., bromobonil,bromoxynil, chloroxynil, dichlobenil, iodobonil, ioxynil andpyraclonil); organophosphorus herbicides (e.g., amiprofos-methyl,anilofos, bensulide, bilanafos, butamifos, 2,4-DEP, DMPA, EBEP,fosamine, glufosinate, glyphosate and piperophos); phenoxy herbicides(e.g., bromofenoxim, clomeprop, 2,4-DEB, 2,4-DEP, difenopenten, disul,erbon, etnipromid, fenteracol and trifopsime); phenoxyacetic herbicides(e.g., 4-CPA, 2,4-D, 3,4-DA, MCPA, MCPA-thioethyl and 2,4,5-T);phenoxybutyric herbicides (e.g., 4-CPB, 2,4-DB, 3,4-DB, MCPB and2,4,5-TB); phenoxypropionic herbicides (e.g., cloprop, 4-CPP,dichlorprop, dichlorprop-P, 3,4-DP, fenoprop, mecoprop and mecoprop-P);phenylenediamine herbicides (e.g., dinitramine and prodiamine);picolinic acid herbicides (e.g., aminopyralid, clopyralid and picloram);pyrazolyl herbicides (e.g., benzofenap, pyrazolynate, pyrasulfotole,pyrazoxyfen, pyroxasulfone and topramezone); pyrazolylphenyl herbicides(e.g., fluazolate and pyraflufen; pyridazine herbicides such ascredazine, pyridafol and pyridate); pyridazinone herbicides (e.g.,brompyrazon, chloridazon, dimidazon, flufenpyr, metflurazon,norflurazon, oxapyrazon and pydanon); pyridine herbicides (e.g.,cliodinate, dithiopyr, fluoroxypyr, haloxydine, picolinafen, pyriclor,thiazopyr and triclopyr); pyrimidinediamine herbicides (e.g., iprymidamand tioclorim); quaternary ammonium herbicides (e.g., cyperquat,diethamquat, difenzoquat, diquat, morfamquat and paraquat);pyrimidinyloxybenzoic acid herbicides (e.g., bispyribac andpyriminobac); thiocarbamate herbicides (e.g., butylate, cycloate,di-allate, EPTC, esprocarb, ethiolate, isopolinate, methiobencarb,molinate, orbencarb, pebulate, prosulfocarb, pyributicarb, sulfallate,thiobencarb, tiocarbazil, triallate and vemolate); sulfonamideherbicides (e.g. asulam, carbasulam, fenasulam, oryzalin, penoxsulam,pyroxsulam); triazine herbicides (e.g., dipropetryn, triaziflam andtrihydroxytriazine, atrazine, chlorazine, cyanazine, cyprazine,eglinazine, ipazine, mesoprazine, procyazine, proglinazine, propazine,sebuthylazine, simazine, terbuthylazine and trietazine, atraton,methometon, prometon, secbumeton, simeton and terbumeton; ametryn,aziprotryne, cyanatryn, desmetryn, dimethametryn, methoprotryne,prometryn, simetryn and terbutryn); triazinone herbicides (e.g.,ametridione, amibuzin, hexazinone, isomethiozin, metamitron andmetribuzin); triazolopyrimidine herbicides (chloransulam, diclosulam,florasulam, flumetsulam, metosulam); urea herbicides (e.g.,benzthiazuron, cumyluron, cycluron, dichloralurea, diflufenzopyr,isonoruron, isouron, methabenzthiazuron, monisouron and noruron;anisuron, buturon, chlorbromuron, chloreturon, chlorotoluron,chloroxuron, daimuron, difenoxuron, dimefuron, diuron, fenuron,fluometuron, fluothiuron, isoproturon, linuron, methiuron, methyldymron,metobenzuron, metobromuron, metoxuron, monolinuron, monuron, neburon,parafluoron, phenobenzuron, siduron, tetrafluoron and thidiazuron;amidosulfuron, azimsulfuron, bensulfuron, chlorimuron, cyclosulfamuron,ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron,foramsulfuron, halosulfuron, imazosulfuron, mesosulfuron, nicosulfuron,orthosulfamuron, oxasulfuron, primisulfuron, pyrazosulfuron,rimsulfuron, sulfometuron, sulfosulfuron and trifloxysulfuron;chlorsulfuron, cinosulfuron, ethametsulfuron, iodosulfuron, metsulfuron,prosulfuron, thifensulfuron, triasulfuron, tribenuron, triflusulfuronand tritosulfuron; buthiuron, ethidimuron, tebuthiuron, thiazafluoronand thidiazuron), etc.

The composition may further comprise an additional fungicidal agent suchas myclobutanil, and fenhexamide, azoxystrobin, azoxystrobincombination, boscalid, bacillus subtilis, copper sulfate,chlorothalonil, copper hydroxide, cymoxanil, dimethomorph,dechloropropene, fosetyl-aluminum, fludioxonil, fenamidone, iprodione,mefenoxam, mancozeb, metalaxyl, metam sodium, potassium bicarbonate,pyraclostrobin, propiconazole, propicocarb, thiram, thiabendazole,thiophanate-methyl, trifloxystrobin, vinclozolin, sulfur, ziram. Theyalso include the following antibacterial agents such as streptomycin andoxytetracycline.

The composition and method of the present invention will be furtherillustrated in the following, non-limiting examples. The examples areillustrative of various embodiments only and do not limit the claimedinvention regarding the materials, conditions, weight ratios, processparameters and the like recited herein.

EXAMPLE

In the instant Example, results are disclosed showing that the methanolextract of dry long pepper (Piper longum L.) fruits are phytotoxicagainst bib lettuce (Lactuca sativa, L.) seedlings. The extract wassubjected to bioassay-guided isolation and yielded one active compound.The active compound was identified as a known compound, calledsarmentine and is the first time it is isolated from P. longum.Phytotoxic activity of sarmentine was examined with a variety ofseedlings from crops, sedge and weeds. Results indicated that sarmentineis a contact herbicide and possesses broad-spectrum herbicidal activity.Sarmentine in the different dried P. longum fruits was quantified byliquid chromatography-mass spectrometry and found to vary dramatically,ranging from almost zero to 0.57%. In addition, a series of sarmentineanalogs were then synthesized to study the structure-activityrelationship (SAR). Results from SAR study suggested that either thelong unsaturated fatty acid (i.e., 2E,4E-decandienoic acid) or the amine(i.e., pyrrolidine) was crucial for activity, but the amide bond with asecondary amine seemed to be necessary for the phytotoxic activity ofsarmentine.

Materials and Methods

Chemical:

Sepra C18-E (50 μM and 60 Å) and Silica gel sorbent (70-230 mesh size)were purchased from Phenomenex® (Torrance, Calif., USA) and FisherScientific (Fair Lawn, N.J., USA), respectively. Polyoxyethylene (20)Sorbitan Monolaurate (i.e., Glycosperse O-20 KFG) and sodium laurylsulfate (i.e., SLS) were obtained as free samples from Lonza Inc.(Allendale, N.J., USA) and Spectrum Chemical Mfg. Corp. (New Brunswick,N.J., USA), respectively. Cyclopentylamine, trans-cinnamic acid, ethyltrans-2-cis-4-decadienoate, hexamethyleneimine, trans-2-decenoic acid,decanoic acid, 4-dimethylaminopyridine are purchased from ACROS Organics(Morris Plains, N.J., USA). All other chemicals were of reagent grades.

Fruits of P. longum and Pre-Treatment:

Four samples of dried P. longum fruits were purchased from Chinesemedicinal herb stores. The fruits were completely ground with a coffeegrinder (Toastmaster Inc., Boonville, Mich., USA). The freshly groundpowder of fruits was extracted with appropriate solvents.

Weed, Sedge and crop Seedlings:

All weed, sedge and crop seedlings were planted in 8 cm×8 cm×7.2 cm or10 cm×10 cm×9 cm plastic pots. Seedlings including pigweed (Amaranthusretroflexus, L.), barnyard grass (Echinochloa crusgalli L.), bindweed(Convolvulus arvensis L.), crabgrass (Digitaria sanguinalis L.),dandelion (Taraxacum officinale F.), lambsquarters (Chenopodium albumL.), bluegrass (Poa annua L.), wild mustard (Brassica kaber L.), blacknightshade (Solanum nigrum L.), curly dock (Rumex crispus L.), horseweed(Conyza Canadensis L.), sweet corn (Zea may S.) and wheat PR 1404(Triticum aestivum L.) were planted in Super Soil potting soil.Seedlings including rice M-104 (Oryza saliva L.), sedge (Cyperusdifformis L.) and sprangletop (Leptochloa fascicularis Lam) were plantedin mud which was collected from rice field (Woodland, Calif., USA). Whentreated, all seedlings were 15 days old except for rice (10 days), wheat(20 days), corn (20 days) sprangletop (20 days old), sedge (20 days old)and horse weed (70 days old).

Bioassay-Guided Extraction and Isolation:

The active compound was isolated by four major steps described asfollows: 1) the methanol extract of freshly ground P. longum fruitpowder was screened in a 96-well plate bioassay with bib lettuce(Lactuca sativa) seedlings; 2) the methanol extract (0.5 gram) wassubjected to a reverse C-18 column and was eluted with 20%, 40%, 60%,80% and 100% methanol in water; Fractions were dried under vacuum andefficacy was re-evaluated by 96-well plate bioassay with bib lettuce(Lactuca sativa) seedlings. The active fraction was used to guide nextstep for separation; 3) Ethyl acetate extract (17.6 g) was loaded into aflash column. The column was sequentially eluted with hexane (1 L),hexane/ethyl acetate (3:1, 1 L), hexane/ethyl acetate (1:1, 1 L), ethylacetate (1 L) and acetone (1 L). Based on thin layer chromatography(TLC), nine fractions were collected. The efficacy of each fraction wasevaluated by foliar spraying of barnyard grass (Echinochloa crusgalli).The concentration of each fraction was 5 mg/mL with a carrier solutionconsisting of 4% ethanol and 0.2% glycosperse O-20 KFG. The activefractions (3.4 g) were combined together and subjected to the next step;4) A secondary silica column was performed with a combination of hexaneand ethyl acetate (3:1) as an elution solvent. The active ingredient(0.96 g) was re-crystallized at −20° C. in a mixture of hexane and ethylacetate and yielded a pure active compound. Purity was examined byliquid chromatography and mass spectrometry (LC/MS). Detailed conditionsfor LC/MS was described later in Materials and Methods.

Structural Elucidation:

Structural identification of the active compound was based on data fromboth Nuclear Magnetic Resonance (NMR) spectra and high resolution massspectrometry. NMR spectra including ¹H, ¹³C, DEPT, COSY, HMQC and HMBCwere acquired from a Bruker Avance 600 spectrometer (Bruker BioSpinCorporation, Billerica, Mass., USA). Chemical shift values are given inppm downfield from an internal standard (trimethylsilane). Signalmultiplicities are represented as singlet (s), doublet (d), doubledoublet (dd), triplet (t), quartet (q), quintet (quint) and multiplet(m). Exact mass of the active compound was determined byhigh-performance liquid chromatography—tandem mass spectroscopy

Comparison of the Active Compound from Different Fruit Samples:

Ground fruit powder (10 g) of different samples was soaked in ethylacetate (50 mL) for 20 h at room temperature. The solution was filteredby a Whatman® qualitative filter paper (NO 1, Ø 155 mm). The residue andfilter paper was washed by ethyl acetate (25 mL). The combined organicphase was dried under vacuum. The weight of each extract was recorded.The active compound in the extracts was quantified by LC/MS.

Quantification of the Active Compound in the Ethyl Acetate Extract ofDried Fruits by (LC/MS):

Chromatographic separation was performed at 25° C. on a Thermo highperformance liquid chromatography (HPLC) instrument equipped withFinnigan Surveyor PDA plus detector, autosampler plus, MS pump and a 4.6mm×100 mm Luna C18 5 μm column (Thermo Electron Corp., San Jose,Calif.). The solvent system consisted of water (solvent A) andacetonitrile (solvent B). The mobile phase began at 10% solvent B andwas linearly increased to 100% solvent B over 20 min and then kept for 4min, and finally returned to 10% solvent B over 3 min and kept for 3min. The flow rate was 0.5 mL/min. The injection volume was 10 μL andthe samples were kept at room temperature in an auto sampler. The activecompound was detected by a positive electrospray ionization mode in afull scan mode (m/z 100-1500 Da) on a LCQ DECA XP^(plus) MassSpectrometer (Thermo Electron Corp., San Jose, Calif.). The flow rate ofthe nitrogen gas was fixed at 30 and 15 arb for the sheath and aux/sweepgas flow rate, respectively. Electrospray ionization (ESI) was performedwith a spray voltage set at 5000 V and a capillary voltage at 35.0 V.The capillary temperature was set at 400° C.

The standard of the active compound was obtained by repeatedcrystallization in laboratory. A series of standard concentrations (125,62.5, 31.25, 15.6, 7.8, 3.9, 1.95 and 0.976 ng/ml) was made in ethanol.Three independent samples (2.5 μg/mL in ethanol) for each extract weremade. Mass spectra were run in a SIM mode with a mass range of221.0-224.0 and retention time (16.94 min). The limits of detection(LOD) of the active compound were determined by running decreasingamounts of standard solution until the ratio of the signal of the activecompound over the background was greater or equal to 3. Theconcentration of fruit samples was presented by an average of threeindependent samples with a standard deviation.

Synthesis of Sarmentine Analogs:

To the ice-cooled carboxylic acid (3 mmole) solution in dichloromethane(20 ml) was sequentially added1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (3.3 mmole) and4-dimethylaminopyridine (3 mmole). After 5 min, amine (3.3 mmole) wasadded in the reaction solution. The reaction was slowly warmed to theroom temperature and lasted overnight. The reaction was extracted withethyl acetate (200 mL). The organic phase was dried with anhydroussodium sulfate. After evaporation under vacuum, the residue was runthrough a silica gel column with an appropriate ratio of ethyl acetatein hexane. The yield of the final products ranged from 85% to 90%. Thefinal products were characterized with proton NMR.

N-Cyclopentylcinnamamide (7)

¹H NMR (CDCl₃): δ (ppm) 7.62 (d, J=15.6 Hz, 1H), 7.50 (d, J=7.0 Hz, 2H),7.35 (m, 3H), 6.37 (d, J=15.6, 1H), 5.61 (d, J=5.0, Hz, 1H, NH), 4.35(sextet, J=7.0, 1H), 2.06 (m, 2H), 1.71 (m, 2H), 1.64 (m, 2H), 1.46 (m,2H).

N-(trans-Cinnamoyl)pyrrolidine (11)

¹H NMR (CDCl₃): δ (ppm) 7.70 (d, J=15.5 Hz, 1H), 7.53 (d, J=7.0 Hz, 2H),7.36 (m, 3H), 6.74 (d, J=15.5, 1H), 3.63 (t, J=7.0, 2H), 3.60 (t, J=7.0,2H), 2.01 (quintet, J=7.0, 2H), 1.91 (quintet, J=7.0, 2H).

N-(trans-Cinnamoyl)piperidine (15)

¹H NMR (CDCl₃): δ (ppm) 7.64 (d, J=15.5 Hz, 1H), 7.52 (d, J=7.2 Hz, 2H),7.36 (m, 3H), 6.90 (d, J=15.5, 1H), 3.67 (s, 2H), 3.59 (s, 2H), 1.68 (m,2H), 1.62 (m, 4H).

N-(trans-Cinnamoyl) hexamethyleneimine (19)

¹H NMR (CDCl₃): δ (ppm) 7.70 (d, J=15.4 Hz, 1H), 7.52 (d, J=7.6 Hz, 2H),7.36 (m, 3H), 6.88 (d, J=15.4, 1H), 3.63 (t, J=6.0, 2H), 3.61 (t, J=6.0,2H), 1.76 (m, 4H), 1.59 (m, 4H).

N-Cyclopentyldecanamide (4)

¹H NMR (CDCl₃): δ (ppm) 5.35 (br, 1H), 4.22 (sextet, J=7.00, 1H), 2.12(t, J=7.20, 2H), 1.98 (m, 2H), 1.59-1.67 (m, 6H), 1.26-1.36 (m, 14H),0.88 (t, J=7.00, 3H).

N-(Decanoyl)pyrrolidine (8)

¹H NMR (CDCl₃): δ (ppm) 3.45 (t, J=6.80, 2H), 3.40 (t, J=6.80, 2H), 2.24(t, J=7.20, 2H), 1.94 (quintet, J=6.80, 2H), 1.84 (quintet, J=6.80, 2H),1.62 (quintet, J=7.20, 2H), 1.25-1.30 (m, 12H), 0.87 (t, J=7.20, 3H).

N-(Decanoyl)piperidine (12)

¹H NMR (CDCl₃): δ (ppm) 3.55 (t, J=5.20, 2H), 3.39 (t, J=5.20, 2H), 2.31(t, J=7.60, 2H), 1.58-1.65 (m, 4H), 1.52-1.57 (m, 4H), 1.20-1.30 (m,12H), 0.87 (t, J=7.20, 3H).

N-(Decanoyl)hexamethyleneimine (16)

¹H NMR (CDCl₃): δ (ppm) 3.52 (t, J=6.00, 2H), 3.42 (t, J=6.00, 2H), 2.30(t, J=7.80, 2H), 1.66-1.74 (m, 4H), 1.60-1.66 (m, 2H), 1.50-1.6.0 (m,4H), 1.20-1.30 (m, 12H), 0.87 (t, J=7.20, 3H).

N-Cyclopentyldecanamide (5)

¹H NMR (CDCl₃): δ (ppm) 6.82 (dt, J₁=15.20, J₂=7.20, 1H), 5.71 (d,J=15.20, 1H), 5.33 (br, 1H), 4.27 (sextet, J=7.00, 1H), 2.15 (m, 2H),2.10 (m, 2H), 1.67 (m, 2H), 1.60 (m, 2H), 1.40 (m, 4H), 1.28 (m, 8H),0.88 (t, J=7.00, 3H).

N-(Decenoyl)pyrrolidine (9)

¹H NMR (CDCl₃): δ (ppm) 6.90 (dt, J₁=15.20, J₂=7.00, 1H), 6.07 (d,J=15.20, 1H), 3.52 (t, J=6.30, 2H), 3.50 (t, J=6.30, 2H), 2.19 (m, 2H),1.96 (quintet, J=7.00, 2H), 1.85 (quintet, J=7.00, 2H), 1.44 (m, 2H),1.28 (m, 8H), 0.88 (t, J=7.00, 3H).

N-(Decenoyl)piperidine (13)

¹H NMR (CDCl₃): δ (ppm) 6.82 (dt, J₁=15.20, J₂=7.00, 1H), 6.23 (d,J=15.20, 1H), 3.59 (t, J=6.30, 2H), 3.47 (t, J=6.30, 2H), 2.17 (m, 2H),1.64 (quintet, J=5.60, 2H), 1.56 (quintet, J=5.60, 4H), 1.44 (quintet,J=7.00, 2H), 1.28 (m, 8H), 0.88 (t, J=7.00, 3H).

N-(Decenoyl)hexamethyleneimine (17)

¹H NMR (CDCl₃): δ (ppm) 6.91 (dt, J₁=15.20, J₂=7.00, 1H), 6.21 (d,J=15.20, 1H), 3.57 (t, J=6.00, 2H), 3.49 (t, J=6.00, 2H), 2.17 (m, 2H),1.73 (m, 4H), 1.56 (m, 4H), 1.45 (m, 2H), 1.28 (m, 8H), 0.88 (t, J=7.00,3H).

N-Cyclopentyl 2E,4Z-decadienamide (6)

¹H NMR (CDCl₃): δ (ppm) 7.55 (dd, J₁=14.80, J₂=11.70, 1H), 6.06 (t,J=11.70, 1H), 5.79 (d, J₁=14.80, 1H), 5.75 (m, 1H), 5.50 (br, 1H), 4.30(sextet, J=7.00, 1H), 2.29 (quartet, J=8.20, 2H), 2.01 (m, 2H), 1.68 (m,2H), 1.61 (m, 2H), 1.40 (m, 2H), 1.28 (m, 6H), 0.88 (t, J=7.00, 3H).

(2E,4Z-Decadienoyl)pyrrolidine (10)

¹H NMR (CDCl₃): δ (ppm) 7.62 (dd, J₁=14.60, J₂=11.70, 1H), 6.17 (d,J=14.60, 1H), 6.13 (t, J=11.70, 1H), 5.78 (m, 1H), 3.55 (t, J=7.00, 2H),3.52 (t, J=7.00, 2H), 2.30 (quartet, J=7.40, 2H), 1.97 (quintet, J=7.40,2H), 1.87 (quintet, J=7.40, 2H), 1.40 (quintet, J=7.40, 2H), 1.29 (m,4H), 0.88 (t, J=7.00, 3H).

(2E,4Z-Decadienoyl)piperidine (14)

¹H NMR (CDCl₃): δ (ppm) 7.59 (dd, J₁=14.60, J₂=11.70, 1H), 6.34 (d,J=14.60, 1H), 6.13 (t, J=11.70, 1H), 5.78 (m, 1H), 3.62 (t, J=7.00, 2H),3.45 (t, J=7.00, 2H), 2.31 (quartet, J=7.40, 2H), 1.67 (quintet, J=7.40,4H), 1.57 (quintet, J=7.40, 2H), 1.40 (m, 2H), 1.27 (m, 4H), 0.88 (t,J=7.00, 3H).

(2E,4Z-Decadienoyl)hexamethyleneimine (18)

¹H NMR (CDCl₃): δ (ppm) 7.64 (dd, J₁=14.60, J₂=11.70, 1H), 6.30 (d,J=14.60, 1H), 6.16 (t, J=11.70, 1H), 5.78 (m, 1H), 3.60 (t, J=7.00, 2H),3.51 (t, J=7.00, 2H), 2.31 (quartet, J=7.40, 2H), 1.76 (m, 4H), 1.57 (m,4H), 1.40 (m, 2H), 1.30 (m, 4H), 0.88 (t, J=7.00, 3H).

Evaluation of Herbicidal Activity:

Herbicidal activity of the active compound and synthesized compounds wasevaluated by foliar spraying. Spraying carrier solution contained 2%ethanol, 0.2% glycosperse O-20 and 0.1% sodium lauryl sulfate. Freshlymade solution with the evaluated compound at a concentration of 5 mg/mLwas used. In the spectrum study, spraying volume was dependent on thefoliar surface area, ranging from approximately 1 to 3 mL/pot plants.One or two pots of plants were treated for each. Number of pots wasdependent on both foliar area and availability. In thestructure-activity relationship study, one pot of barnyard grass wasused and the spraying volume of each compound was 3 mL of 5 mg/mL.Phytotoxic activity was evaluated at least 3 days after spraying.Efficacy of phytotoxic activity was graded as I (no effect), II (<20%),III (20-40%), IV (40-60%), V (60-80%) and VI (80-100%).

Results

Bio-Assay Guided Purification:

The active compound was obtained by the following procedures. Initialexperiment indicated phytotoxic activity in the methanol extract of P.longum dried fruits. The active ingredient(s) existed in the fractionwith 100% methanol elution when a reversed C18 column was used, and nophytotoxic activity was shown from other fractions at 5 mg/mL (notshown). In our next experiment, ethyl acetate extract was run though aflash silica column. The elution was collected as 9 fractions (based onTLC indication). The weight of each fraction from 1 to 9 was 3.0, 1.0,1.7, 3.6, 0.9, 2.5, 1.2, 0.2 and 2.3 gram, respectively. Two out of ninefractions were active against barnyard grass (FIG. 1). The combined twoactive fractions (i.e., Fr 5 & Fr 6, 3.4 g) were re-run through a silicacolumn and yielded slightly yellowish oil as the active compound. Thisoil was re-crystallized in a mixture of hexane and ethyl acetate at −20°C. and obtained as colorless oil (0.83) at room temperature.

Structure Elucidation:

The active compound was identified based on the following evidence: Highresolution mass data (TOF MS ESI⁺) is 222.5386, indicating that themolecular formula of the active compound is most possibly C₁₄H₂₃NO. Thedata from ¹H and ¹³C NMR listed in the Table 1 further supported thismolecular formula. Data in the proton NMR indicate that there are atotal of 23 protons including 4-olefenic protons (4CH), 1CH₃ and 8CH₂.Data from ¹³C NMR and DEPT-135 (not shown) confirmed that there are 14carbons including 1 amide carbonyl (N—CO—), 4CH, 8CH₂ and 1CH₃. Datafrom COSY (not shown) indicated that 2 spin systems are present in themolecule. One contains 4 consecutive CH₂. And another contains 4conjugated olefin protons which are further connected to CH₂CH₂CH₂CH₃.Moreover, data from HMBC (not shown) indicated that the above-mentioned2-spin systems are connected through an amide carbonyl to give the planestructure. Finally, geometric conformation is trans for both doublebonds based on coupling constants (J=14.4 Hz). This active compound(Table 1) is a known compound called sarmentine, which was firstpurified from dried P. sarmentosum fruit powder (Likhitwitayawuid, K.,Ruangrungsi, N, Lange, G and Decicco, C., Structural Elucidation andSynthesis of New Components isolated from Piper Samentosum, Tetrahedron1987 (43) 3689-3694) and also from P. nigrum (Kiuchi, F.; Nakamura, N.;Tsuda, Y.; Kondo, K.; Yoshimura, H. Studies on crude drugs effective onvisceral larva migrans, IV isolation and identification of larvicidalprinciples in Pepper, Chem. Pharm. Bull, 1988, 36, 2452-2465). However,it is the first time that it was isolated from P. longum and found to bea phytotoxic compound.

Table 1 ¹H and ¹³C NMR (CDCl₃) data of sarmentine

NO ¹³C NMR data ¹H NMR data  1 165.4 —  2 120.0 6.08 1H d (J = 14.4 Hz) 3 142.4 7.26 1H dd (J₁ = 14.4, J₂ = 10.8 Hz)  4 128.8 6.16 1H dd (J₁ =10.8, J₂ = 9.6 Hz)  5 143.4 6.07 1H dt (J₁ = 14.4, J₂ = 7.2 Hz)  6 33.12.13 2H quartet (J = 7.2 Hz)  7 26.3 1.39 2H quintet (J = 7.2 Hz)  824.5 1.27 2H m  9 22.7 1.27 2H m 10 14.2 0.87 3H t (J = 7.2 Hz)  1′ 46.63.50 2H t (J = 7.2 Hz)  2′ 31.5 1.95 2H quintet (J = 7.2 Hz)  3′ 28.61.85 2H quintet (J = 7.2 Hz)  4′ 46.0 3.52 2H t (J = 7.2 Hz)

Herbicidal Spectrum of Sarmentine:

Phytotoxic activity of sarmentine depended on the concentration. Theoptimal concentration of sarmentine was 5 mg/mL for excellent control ofbarnyard grass (FIG. 2). Therefore, such a concentration was subjectedto herbicidal spectrum study. Due to the hydrophobic property ofsarmentine, a carrier solution containing 0.2% glycosperse O-20, 2%ethanol and 0.1% sodium lauryl sulfate was used. Although this solutioncontains a high concentration of surfactants, sarmentine can staysuspended in this solution no more than 15 min. Therefore, sarmentinewas made freshly just before spraying. In addition, this solution didnot show any phytotoxic activity toward tested plants when results wererecorded.

Sarmentine displayed phytotoxic activity against a variety of plantsincluding sedge, crops and weeds (Table 2).

TABLE 2 Phytoxtoxic activity of sarmentine toward different plants NoPlant name Efficacy* 1 Pigweed VI (Amaranthus retroflexus, L.) 2Barnyard grass VI (Echinochloa crusgalli L.) 3 Bindweed VI (Convolvulusarvensis, L.) 4 Crabgrass VI (Digitaria sanguinalis L.) 5 Horse weed II(Conyza Canadensis L.) 6 Sedge III (Cyperus difformis L.) 7 SprangletopVI (Leptochloa fascicularis Lam.) 8 Dandelion VI (Taraxacum officinaleF.) 9 Lambsquarters VI (Chenopodium album L.) 10 Bluegrass VI (Poa annuaL.) 11 Wild mustard VI (Brassica kaber L.) 12 Black nightshade VI(Solanum nigrum L.) 13 Curly dock VI (Rumex crispus L.) 14 Sweet corn VI(Zea mays S.) 15 Wheat (PR 1404) VI (Triticum aestivum L.) 16 Rice (M104) I (Oryza saliva L) *Efficacy of phytotoxic activity was graded as I(no effect), II (<20%), III (20-40%), IV (40-60%), V (60-80%) and VI(80-100%).

Phytotoxic activity of sarmentine was dependent on the plants, rangingfrom zero to 100% killing. No visible phytotoxic activity (after 10days) was shown on rice plants toward sarmentine. Slightly phytotoxicactivity of sarmentine toward sedge and horseweed was shown. However,highly phytotoxic activity of sarmentine was displayed toward pigweed,barnyard grass, bindweed, crabgrass, sprangletop, dandelion,lambsquarters, blue grass, wild mustard, black nightshade, curly dock,sweet corn and wheat. The phytotoxic activity may be related to age ofplants. For example, horseweed (70-day-old) was much older than otherweeds, but less phytotoxic activity was shown.

Structure-Activity Relationship (SAR):

The SAR study suggested that either the long unsaturated fatty acid orpyrrolidine of sarmentine is crucial for phytotoxic activity, but theamide bond with a secondary amine seemed to be necessary. Thisconclusion was supported by the following experimental results which aresummarized below in Table 3.

TABLE 3 Phytotoxic activity of sarmentine and its analogs towardbarnyard grass No Structures Efficacy * 1 vector I 2

VI 3

II 4

II 5

II 6

II 7

II 8

VI 9

VI 10

VI 11

VI 12

VI 13

VI 14

VI 15

IV 16

VI 17

VI 18

VI 19

II 20

V 21

VI 22

VI 23

I 24

I 25

I 26

I * Efficacy of phytotoxic activity was graded as I (no effect), II(<20%), III (20-40%), IV (40-60%), V (60-80%) and VI (80-100%).

Phytotoxic activity remained the same as the acid moiety of sarmentinewas replaced by structurally similar fatty acids such as2E,4Z-decandienoic acid (i.e., geometric isomer of 2E,4E-decandienoicacid) with two double bonds (10), 2E-decenoic acid with one double bond(9) and decanoic acid without any double bond (8), and even structurallydifferent acid such as trans-cinnamic acid (11). This suggested that theacid moiety of sarmentine can be various acids when the amine ispyrrolidine. Similarly, when the acid moiety remained the same, theamine can be various. For example, phytotoxic activity remained the samein terms of decanoic acid when the amine was changed from a five memberring (8) to a six or seven member ring (12 & 16, respectively). However,phytotoxic activity dropped dramatically when the amide bond with asecondary amine was changed into an ester bond (e.g., 3) and an amidebond with a primary amine (e.g., 4-7). In addition, results from SARstudy (Table 3) also indicated that the amine moiety of sarmentine,pyrrolidine (24), and its analogs such as cyclopentylamine (23),hexamethyleneimine (26) and piperidine (25) were non toxic to barnyardgrass; but the analogs of the acid moiety of sarmentine such as decanoicacid (21), 2E-decenoic acid and (22) and trans-cinnamic acid (20) werevery active. To obtain a better SAR, the length of carbon chain in theacid moiety and disubstituted amines (non ring system) should be furtherinvestigated.

Symptom of Phytotoxic Activity:

Sarmentine is a contact phytotoxic compound. When plants were treatedwith sarmentine solution, slightly black tiny spots would first show onthe leaves and then became bigger and bigger until they covered thewhole leaves, especially obvious on the barnyard grass. This symptomcould be observed within half an hour to 2 hours after spraying.Predominant phytotoxic activity happened within 48 h after spraying.This symptom was observed very similarly to that of middle-chain fattyacids such as decanoic acid (21, Table 4) and 2E-decenoic acid (22,Table 3) during the SAR study.

Quantification of Sarmentine in Different P. longum Fruit Samples:

The standard curve of sarmentine (Y=83324X-30784, R²=0.9998; X and Ystand for the concentration of sarmentine and peak area, respectively)was obtained. The limit of detection for sarmentine was 12 pg perinjection (or 1.2 ng/mL). Based on this external standard curve, theconcentration of sarmentine in the extracts of four different P. longumfruit samples varied from 0.0005% to 0.57%. These results are shown inTable 4.

TABLE 4 The concentration of sarmentine in different samples of dry P.lungum fruits Extract weight Percent of (g) from Percent sarmentine 10gram of content of in the ethyl ground dried sarmentine Sample acetateextract fruit powder in the dry fruit 1 12.66 ± 0.45  0.45 ± 0.03 0.5697± .0380  2 1.83 ± 0.10 0.51 ± 0.03 0.0933 ± 0.0055 3  0.01 ± 0.001 0.55± 0.02 0.00055 ± 0.00002 4 0.008 ± 0.001 0.66 ± 0.03 0.00053 ± 0.00002

This difference in amounts of sarmentine present in each of the samplesmay result from the age and/or origin of the dried fruits. The quantityof sarmentine in the ground powder of dried P. longum fruits can beviewed by naked eyes because the higher the concentration of sarmentineis, the more oily the ground powder is. For example, no oil was visiblefor the ground powder of samples 3 and 4, but oily powder was seen inthe ground powder from samples 1 and 2.

Although this invention has been described with reference to specificembodiments, the details thereof are not to be construed as limiting, asit is obvious that one can use various equivalents, changes andmodifications and still be within the scope of the present invention.

Various references are cited throughout this specification, each ofwhich is incorporated herein by reference in its entirety.

What is claimed is:
 1. A method for controlling monocotyledonous, sedgeor dicotyledonous weeds in a substrate comprising applying to the weedsand/or substrate an amount of sarmentine effective to controlmonocotyledonous or dicotyledonous weeds in said substrate.
 2. Themethod according to claim 1, wherein said weeds are broadleaved and/orgrass weeds.
 3. The method according to claim 1, wherein said substrateis soil, sediment, artificial growth substrates or water.
 4. The methodaccording to claim 1, wherein said weed is a rice weed.
 5. The methodaccording to claim 1, where said sarmentine is applied to the substrateprior to emergence of said weed.
 6. The method according to claim 1,wherein said sarmentine is obtainable from a member of the Piper speciesin the Piperaceae family.
 7. The method according to claim 1, furthercomprising applying one or more other herbicidal agents.